Molecular imaging techniques have become indispensable for diagnosing diseases early, before they cause anatomical changes, and for selection and monitoring of treatment. Current clinical and in vivo molecular imaging modalities offer high molecular sensitivity but suffer from low spatial resolution. In this proposal, we will investigate the combination of two emerging x-ray based imaging techniques for high-sensitivity and high- resolution molecular imaging. The goal of the proposal is to design, build, and thoroughly investigate an in vivo x-ray based molecular imaging system. My preliminary simulation data indicate that x-ray fluorescence computed tomography (XFCT) has significantly higher sensitivity than conventional x-ray imaging for visualizing molecular probes containing high atomic number elements, such as gold and platinum. The proposed design combines the high molecular sensitivity of XFCT with the ability of x-ray spectral CT (XSCT) to visualize the anatomy multispectrally. The XFCT/XSCT system should advance our ability to develop new imaging probes and therapeutic drugs for a wide range of diseases. The course of the proposed research begins with computer simulations that will determine the optimal design of clinical and preclinical XFCT/XSCT scanners. In the second part of the proposed research, I will build an in vivo microXFCT/XSCT scanner and its performance will be evaluated through a series of phantom experiments. In the last step, biodistribution of novel gold nanoparticles with a number of surface chemistries will be determined in vivo. The system when fully developed and disseminated can study fundamental biological processes and augment existing tools such as PET and optical imaging. To ensure the success of the proposed project, I have assembled a strong team of physicists, biologists, electrical engineers, and chemist that will work together towards achieving the goals of the project. During the mentored K99 award period, I will receive training in medical imaging physics, experimental experience, as well as training in molecular imaging techniques. During the independent R00 phase, the skills I acquired during the mentored phase will be applied towards the design and development of an in vivo microXFCT/XSCT scanner. The expertise I will develop during the award period will be critical for the realization of my long-term career goal, which is to thoroughly investigate dose-enhanced radiation therapy (DERT) for improvement of treatment outcomes. Uniform specific tumor uptake of molecular probes containing high atomic number elements is essential in DERT. With the K99/R00 award, I will establish important collaborations that may lead to development of new molecular probes making DERT a feasible treatment modality.